There are numerous situations wherein it may be desirable to control the release of amino-group-containing substances to liquid media. For example, it may be desirable to control the release of an amino-group-containing drug or cytotoxin to a cell population or specific members of a cell population. It may also be desirable to control the cleavage of various cross-linked proteins or peptides, for example, in analyzing the spatial relationships in a complex of large amino-group-containing molecules such as peptides or proteins.
One specific situation in which controlled release is desirable is in delivering a biologically active compound through the cell membrane to inner cell structures, for example, where the compound has a low or reduced effect if trapped in the medium outside the cell membrane but is more potent once released inside the cell.
It is also desirable to be able to deliver biologically active compounds to selected cells in a heterogeneous cell population. For example, in treating diseased or infected cells such as virus-infected cells or transformed or malignant cells, it is desirable to deliver cytotoxins anti-viral agents or growth regulating factors to the diseased or malignant cells but not to normal cells
One approach disclosed for targeting biologically active compounds to malignant cells uses an antibody-toxin conjugate. The antibody is specific for malignant cells and delivers the toxin to them. To be effective, these systems should deliver the toxin with high selectivity to the target cells without unnecessarily reducing the effectiveness of the active substance. These problems are particularly important where the goal is destruction of infected or diseased cells in vivo without harming normal cells.
A variety of protein crosslinking reagents are commercially available. Those crosslinkers in widest use are heterobifunctional reagents using maleimide and N-hydroxysuccinimide esters. Specific coupling of two proteins via a non-cleavable thioether bond is achieved by introducing an SH group into one of the proteins, as disclosed by Kitagawa, U.S. Pat. No. 4,150,033. Commercially available cleavable crosslinkers are limited to disulfide, carboxylic acid ester, and vic-glycol groupings requiring -SH reagents, strong nucleophiles, or periodate oxidation, which permanently modify many proteins, cf. Wang et al., Isr. J. Chem. 12:375-378, 1974; Lutter et al , FEBS 48 288-292, 1974.Disulfide reagents are only slowly cleaved within the cells.
It was found by Neville, CRC Crit. Revs. Ther. Drug Carrier Syst. 2:329-352, 1986, that the efficacy of holo-toxin conjugates coupled via disulfide bonds is not superior to thioether crosslinking.
Additional cleavable bifunctional crosslinking reagents are known, including Lambert et al., J. Mol. Biol. 149: 451-476 (198I) and Wang et al., Isr. J. Chem. 12: 375-389 (I974) wherein are disclosed bifunctional crosslinking reagents containing a cleavable disulfide bond The reagents are used to characterize biochemical systems.
Carlsson et al., Biochem. J. 173: 723-737 (1978) disclose a procedure for forming disulfide bonds between two different proteins using the bifunctional reagent N-succinimidyl-3-(2-pyridyldithio)propionate.
More specifically, certain monoclonal antibodies, toxins, and conjugates thereof are known.
Vitetta et al, in Science 219: 644-650 (1983) and Edwards, Pharmacol. Ther. 23: 147-177 (1983) disclose disulfide-linked conjugates of toxins and monoclonal antibodies specific to cell-surface structures. These conjugates are used to target toxins toward specific cells having surface structures recognized by the antibodies.
Ramakrishnan et al., in Cancer Research 44: 1398-1404 (1984) disclose conjugating pokeweed anti-viral protein (PAP) to anti-Thy 1.1, a monoclonal antibody. The conjugate is used to inhibit protein synthesis selectively in Thy 1.1-positive target leukemia cells. The linker used to form the conjugate is N-succinimidyl-3-(2-pyridyldithio)propionate. When the disulfide bond is cleaved, the free PAP toxin is produced.
Ritz et al., Nature 283: 583-585 (1980) disclose a monoclonal antibody (J5) that is specific for common acute lymphoblastic leukemia antigen.
Stirpe et al. , J. Biol Chem. 255: 6947-6953 (1980) disclose a method for preparing gelonin, a protein cytotoxin.
Barbieri et al., Biochem. J. 203: 55-59 (1982) disclose the purification and partial characterization of an antiviral protein known as pokeweed antiviral proteins ("PAP-S").
Neville et al., U.S. Pat. No. 4,359,457, disclose a conjugate of anti-Thy 1.2 monoclonal antibody and ricin used as a tumor suppressive composition against lymphoma. The linking agent used is m-maleimidobenzoyl-N-hydroxysuccinimide.
The above-described approaches either depend on the toxicity of an antibody-toxin conjugate, or they depend on disulfide bond cleavage, a phenomenon that may be difficult to control temporally and spatially to avoid release of the toxin before delivery to the targeted cells.
An acid cleavable protein crosslinker has been described based on a citraconic anhydride group by Blattler et al., U.S. Pat. Nos. 4,542,225, 4,618,492. Deficiencies in this scheme include the fact that the hydrolysis rate is not linearly correlated with the hydrogen ion concentration (a 30-fold change in hydrolysis for a 600-fold change in [H+], there is a potential for forming an irreversible crosslink through Michael type additions of cellular SH groups to the double bond, there is a failure to demonstrate significant hydrolysis of crosslinked product at the low end of the intravesicle pH (5.4), i.e., &lt;25% hydrolysis at pH 5.5 in 10 hours, and there is a failure to demonstrate efficacy over non-cleavable crosslinkers in a system involving protein uptake either by tissue culture or in vivo, as well as a lengthy synthetic sequence.
Kirby et al. , Proc. Biochem. Soc. Symp. 31: 99-103 (1970) disclose that maleic acid amides are rapidly hydrolyzed below pH 3, and that substitution of maleamic acid increases that rate, with a t-butyl substituent providing the largest increase and a methyl substituent the smallest.
Dixon et al. , Biochem. J. 109: 312-314 (1968) disclose reversible blocking of amino groups using 2-methyl-maleic (citraconic) anhydride as a blocking agent. The amine bond between the citraconyl residue and a lysine residue of insulin was not cleaved at pH 6.5; when the pH was lowered to 3.5 at 20.degree. C. overnight, there was total release of the blocking group, leaving the insulin unchanged.
The concept of a prodrug is not new, and has been described by Albert in Nature 182:421-423, 1975. The acid catalyzed hydrolytic properties of orthoesters, acetals, and ketals are well described, particularly by Cordes et al in Chem. Rev. 74 581-603, 1974, and have been used to achieve prodrugs in the slow release of subcutaneously implanted steroid contraceptives from a solid orthoester polymer matrix, Heller et al. , Polym. Eng. Sci. 21: 727, 1981, and in the gastric release of acetaminophen from a more pleasant tasting acetal prodrug, Hussain, U.S. Pat. No. 3,786,090. The log of the hydrolytic rate constant of this drug was found to be linear with pH over the range of pH 2-6, Hussain et al., J. Pharm. Sci. 67: 546-546 1978.
The extension of the above basic chemistry to a protein crosslinker capable of release within intracellular compartments was not trivial. The starting materials for orthoester synthesis, ketene acetals, are notoriously difficult to work with because of cationic polymerization side reactions. Synthetic routes could not use strong acid conditions, and the desired maleimide functionality limited many approaches, such as orthoesters via the Pinner synthesis. These limitations were particularly bothersome for the synthesis of the heterobifunctional reagents which classically employ harsh conditions
Neville et al., in U.S. Pat. Nos. 4,356,117; 4,359,457; 4,440,747; 4,500,637; 4,520,011; and 4,520,26; elucidate the concepts and utility of constructing monoclonal antibody-protein toxin conjugates (immunotoxins) directed at specific unwanted target cells. Holo-ricin based immunotoxins have greater efficacy than ricin A chain immunotoxins. The enhanced efficiency of such holotoxin conjugates over those constructed with toxin A-chains has been documented. The discrimination between target and nontarget cell was maintained by reversibly blocking the ricin toxin B chain binding site with lactose, since this site has been shown to be essential for full immunotoxin efficacy, as lactose is extruded from the cell following endocytosis. The ricin binding site is required for efficient membrane translocation of ricin within the cell, and lactose blocks ricin binding outside the cell but not inside the cell because lactose is actively transported out of the cell. However, such conjugates reversibly blocked with lactose have limited in vivo efficacy, since high concentrations of lactose cannot be maintained in vivo without untoward effects.
Acid cleavable crosslinkers permit reversible blockade of the ricin binding sites by crosslinking asialoglycoproteins, and asialoglycopeptides, and mannose binding proteins to these sites which can dissociate in the acidified vesicle. In addition, the translocation functions of mutant diphtheria toxins such as CRM 103 and CRM 107, which are partially blocked by coupling to monoclonal antibodies with conventional crosslinkers (Greenfield et al. Science 238 536-539, 1987) can be uncoupled within the cell using a cleavable crosslinker. Since these toxin mutants, which are binding domain mutants, have reduced non-target cell specificity to begin with, they should be suitable for in vivo use Immunotoxins of CRM 103 and CRM 107 constructed with acid cleavable crosslinkers should provide an extra I to 2 logs increase in efficacy over the conventional crosslinkers.
Polyethylene glycol (PEG) has been conjugated to proteins by a variety of procedures to block certain functional domains in vivo, Aubuchowski et al., J. Biol. Chem. 252: 3582-3586, 1976, These PEG conjugates can be used to administer an enzyme protein missing from the body in order to correct an enzyme deficiency disease. PEG coupling can minimize two problems, namely, rapid clearance of the unmodified protein from the vascular system, either antibody or extra antibody mediated, and the formation of antibodies to the foreign protein. Rapid clearance and antigenic stimulation are also problems concerning the in vivo use of immunotoxins. However, PEG, like antibody coupling, also interferes with toxin translocation.